Rapid method for cloning and expression of cognate antibody variable region gene segments

ABSTRACT

In the method as reported herein the isolation of nucleic acid segments encoding antibody variable domains and the insertion of the isolated nucleic acid segments in eukaryotic expression plasmids is performed without the intermediate isolation and analysis of clonal intermediate plasmids. Thus, in the method as reported herein the intermediate cloning, isolation and analysis of intermediate plasmids is not required, e.g. by analysis of isolated transformed  E. coli  cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/310,966, filed Jun. 20, 2014, which is a continuation ofInternational Patent Application No. PCT/EP2012/076155, having aninternational filing date of Dec. 19, 2012, the entire contents of whichare incorporated herein by reference in its entirety, which claimsbenefit under 35 U.S.C. §119 to European Patent Application No.11194861.8, filed Dec. 21, 2011.

SEQUENCE LISTING

The instant application contains a Sequence Listing submitted viaEFS-Web and hereby incorporated by reference in its entirety. Said ASCIIcopy, created on Apr. 3, 2017, is named P30785_US_1_US_Seq_Listing.txt,and is 10,279 bytes in size.

FIELD OF THE INVENTION

Herein is reported a method for the isolation (cloning, expression, andselection) of an antibody starting from a single antibody-producingB-cell, wherein the individual steps of the method are all performed insolution allowing for the identification of antibodies with the desiredspecificity.

BACKGROUND OF THE INVENTION

For obtaining cells secreting monoclonal antibodies the hybridomatechnology developed by Koehler and Milstein is widely used. But in thehybridoma technology only a fraction of the B-cells obtained from animmunized experimental animal can be fused and propagated. The source ofthe B-cells is generally an organ of an immunized experimental animalsuch as the spleen.

Zubler et al. started in 1984 to develop a different approach forobtaining cells secreting monoclonal antibodies (see e.g. Eur. J.Immunol. 14 (1984) 357-363, J. Exp. Med. 160 (1984) 1170-1183). Thereinthe B-cells are obtained from the blood of the immunized experimentalanimal and co-cultivated with murine EL-4 B5 feeder cells in thepresence of a cytokine comprising feeder mix. With this methodology upto 50 ng/ml antibody can be obtained after 10-12 days of co-cultivation.

Weitkamp, J.-H., et al., (J. Immunol. Meth. 275 (2003) 223-237) reportthe generation of recombinant human monoclonal antibodies to rotavirusfrom single antigen-specific B-cells selected with fluorescentvirus-like particles. A method of producing a plurality of isolatedantibodies to a plurality of cognate antigens is reported in US2006/0051348. In WO 2008/144763 and WO 2008/045140 antibodies to IL-6and uses thereof and a culture method for obtaining a clonal populationof antigen-specific B cells are reported, respectively. A culture methodfor obtaining a clonal population of antigen-specific B-cells isreported in US 2007/0269868. Masri et al. (in Mol. Immunol. 44 (2007)2101-2106) report the cloning and expression in E. coli of a functionalFab fragment obtained from single human lymphocyte against anthraxtoxin. A method for preparing immunoglobulin libraries is reported in WO2007/031550.

In WO 2010/056898 rapid expression and cloning of human monoclonalantibodies from memory B-cells is reported. A rapid and efficientsingle-cell manipulation method for screening antigen-specificantibody-producing cells from human peripheral blood is reported by Jinet al. (Jin, A., et al., Nature Medicine 15 (2009) 1088-1093). Lightwoodet al. (Lightwood, D. J., et al., J. Immunol. Meth. 316 (2006) 133-143)report antibody generation through B-cell panning on antigen followed byin situ culture and direct RT-PCR on cells harvested en masse fromantigen-positive wells.

SUMMARY OF THE INVENTION

In the method as reported herein the isolation of nucleic acid fragmentsor segments encoding antibody variable domains (light and heavy chain)and the insertion of the isolated nucleic acid fragments or segments ineukaryotic expression cassettes (one cassette each for the light andheavy chain, respectively) is performed without the intermediateisolation and analysis of clonal intermediate plasmids/cassettes. Thus,in the method as reported herein the intermediate cloning, isolation andanalysis of intermediate plasmids/cassettes is not required, e.g. byanalysis of isolated transformed E. coli cells, thus, resulting in afaster method.

One aspect as reported herein is a method for the isolation of nucleicacids encoding cognate variable domains of an antibody comprising thefollowing steps:

-   -   synthesizing single stranded cDNA using the RNA obtained from an        antibody secreting B-cell as template in an RT-PCR,    -   amplifying the variable domain encoding nucleic acids in a PCR        and thereby isolating the nucleic acid fragments encoding the        cognate variable domains of an antibody,        whereby the PCR primer are removed after the PCR.

In one embodiment the method is performed without the isolation andanalysis of intermediate nucleic acids.

One aspect as reported herein is a method for producing an antibodycomprising the following step:

-   -   cultivating a eukaryotic cell comprising a nucleic acid encoding        an antibody, and    -   recovering the antibody from the cell or the cultivation medium,        whereby the nucleic acid encoding the antibody is obtained by    -   synthesizing single stranded cDNA using the RNA obtained from an        antibody secreting B-cell as template in an RT-PCR,    -   amplifying the variable domain encoding nucleic acid(s) in a        PCR, and    -   inserting the variable domain encoding nucleic acid(s) in one or        more eukaryotic expression plasmids.

In one embodiment the PCR primer are removed after the PCR.

In one embodiment the method is performed without the isolation andanalysis of intermediate nucleic acids.

One aspect as reported herein is a method for producing an antibodycomprising the following step:

-   -   cultivating a eukaryotic cell transfected with one or more        expression plasmids encoding the antibody heavy and light chains        whereby the one or more expression plasmids have been prepared        from a pool of plasmid transformed E. coli cells,    -   recovering the antibody from the cell or the cultivation medium.

One aspect as reported herein is a method for producing an antibodycomprising the following step:

-   -   recovering the antibody from the cultivation medium of a        eukaryotic cell comprising a nucleic acid encoding the antibody,    -   whereby the nucleic acid encoding the antibody is obtained by    -   amplifying cognate variable domain encoding nucleic acids from        single stranded cDNA obtained from the RNA of an antibody        secreting B-cell as template in a PCR, and    -   inserting the variable domain encoding nucleic acids in a        eukaryotic expression plasmid by ligation independent cloning,    -   wherein a pool of nucleic acids encoding the antibody light and        heavy chain variable domain, respectively, is used for the        insertion.

In one embodiment the method comprises as first step:

-   -   synthesizing single stranded cDNA using the RNA obtained from an        antibody secreting B-cell as template.

In one embodiment the PCR primer are removed after the PCR.

In one embodiment the method is performed without the isolation andanalysis of intermediate nucleic acids.

One aspect as reported herein is a method for producing an antibodycomprising the following step:

-   -   cultivating a eukaryotic cell transfected with an expression        plasmid encoding the antibody, whereby the eukaryotic cell has        been transfected with a pool of expression plasmids that has        been prepared from a pool of plasmid transformed E. coli cells,    -   recovering the antibody from the cell or the cultivation medium.

In one embodiment the nucleic acid encoding the antibody is obtained by

-   -   amplifying cognate variable domain encoding nucleic acids from        single stranded cDNA obtained from the RNA of an antibody        secreting B-cell as template in a PCR, and    -   inserting the variable domain encoding nucleic acids in a        eukaryotic expression plasmid by ligation independent cloning.

In one embodiment the method comprises as first step:

-   -   synthesizing single stranded cDNA using the RNA obtained from an        antibody secreting B-cell as template.

In one embodiment the PCR primer are removed after the PCR.

In one embodiment the method is performed without the isolation andanalysis of intermediate nucleic acids.

In one embodiment of all aspects the B-cell is a rabbit B-cell.

In one embodiment of all aspects the B-cell is a single depositedB-cell.

In one embodiment of all aspects the B-cell is cultivated for about 7days.

In one embodiment of all aspects the B-cell and its progeny producesmore than 20 ng/ml antibody in 7 days of co-cultivation with feedercells starting from a single cell.

In one embodiment of all aspects the PCR primer have the nucleic acidsequences of SEQ ID NO: 5 and 6 or SEQ ID NO: 7 or 8.

In one embodiment of all aspects the nucleic acid fragments are insertedinto the expression plasmid by sequence and ligation independentcloning.

In one embodiment of all aspects about 300 ng nucleic acid is used inthe insertion reaction.

In one embodiment of all aspects a pool of nucleic acids is used for theinsertion.

In one embodiment of all aspects the expression plasmid is obtained bysequence and ligation independent cloning of the variable domainencoding nucleic acid into a variable domain free amplified expressionplasmid.

In one embodiment of all aspects the plasmid is linearized prior to theamplification.

DETAILED DESCRIPTION OF THE INVENTION Definitions

“Affinity” refers to the strength of the total sum of non-covalentinteractions between a single binding site of a molecule (e.g., anantibody) and its binding partner (e.g., an antigen). Unless indicatedotherwise, as used herein, “binding affinity” refers to intrinsicbinding affinity which reflects a 1:1 interaction between members of abinding pair (e.g., antibody and antigen). The affinity of a molecule Xfor its partner Y can generally be represented by the dissociationconstant (Kd). Affinity can be measured by common methods known in theart, including those described herein. Specific illustrative andexemplary embodiments for measuring binding affinity are described inthe following.

The term “amino acid” as used within this application denotes the groupof carboxy α-amino acids, which directly or in form of a precursor canbe encoded by a nucleic acid. The individual amino acids are encoded bynucleic acids consisting of three nucleotides, so called codons orbase-triplets. Each amino acid is encoded by at least one codon. This isknown as “degeneration of the genetic code”. The term “amino acid” asused within this application denotes the naturally occurring carboxyα-amino acids comprising alanine (three letter code: ala, one lettercode: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp,D), cysteine (cys, C), glutamine (gln, Q), glutamic acid (glu, E),glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu,L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F),proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp,W), tyrosine (tyr, Y), and valine (val, V).

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-binding activity.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)₂; diabodies; linear antibodies; single-chain antibody molecules(e.g. scFv); single domain antibodies; and multispecific antibodiesformed from antibody fragments.

The “class” of an antibody refers to the type of constant domain orconstant region possessed by its heavy chain. E.g. there are five majorclasses of antibodies in the human: IgA, IgD, IgE, IgG, and IgM, andseveral of these may be further divided into subclasses (isotypes),e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constantdomains that correspond to the different classes of immunoglobulins arecalled α, δ, ε, γ, and μ, respectively.

The term “cognate pair of antibody variable domains” denotes a pair ofantibody variable domains that is obtained from a single antibodysecreting B-cell, i.e. which has been generated as pair during theimmune response of a mammal due to the contact with an immunogenicmolecule or which have been assembled randomly during a displayapproach.

An “effective amount” of an agent, e.g., a pharmaceutical formulation,refers to an amount effective, at dosages and for periods of timenecessary, to achieve the desired therapeutic or prophylactic result.

The term “expression” as used herein refers to transcription and/ortranslation and secretion processes occurring within a cell. The levelof transcription of a nucleic acid sequence of interest in a cell can bedetermined on the basis of the amount of corresponding mRNA that ispresent in the cell. For example, mRNA transcribed from a sequence ofinterest can be quantified by qPCR or RT-PCR or by Northernhybridization (see Sambrook, et al., Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989)). Polypeptides encoded by a nucleic acid can bequantified by various methods, e.g. by ELISA, by assaying the biologicalactivity of the polypeptide, or by employing assays that are independentof such activity, such as Western blotting or radioimmunoassay, usingimmunoglobulins that recognize and bind to the polypeptide (seeSambrook, et al., (1989), supra).

An “expression cassette” denotes a construct that contains the necessaryregulatory elements, such as promoter and polyadenylation site, forexpression of at least the contained nucleic acid in a cell.

The term “expression machinery” denotes the sum of the enzymes,cofactors, etc. of a cell that is involved in the steps of geneexpression beginning with the transcription step of a nucleic acid orgene (i.e. also called “gene expression machinery”) to thepost-translational modification of the polypeptide encoded by thenucleic acid. The expression machinery e.g. comprises the steps oftranscription of DNA into pre-mRNA, pre-mRNA splicing to mature mRNA,translation into a polypeptide of the mRNA, and post translationalmodification of the polypeptide.

An “expression plasmid” is a nucleic acid providing all requiredelements for the expression of the comprised structural gene(s) in ahost cell. Typically, an expression plasmid comprises a prokaryoticplasmid propagation unit, e.g. for E. coli, comprising an origin ofreplication, and a selectable marker, a eukaryotic selection marker, andone or more expression cassettes for the expression of the structuralgene(s) of interest each comprising a promoter, a structural gene, and atranscription terminator including a polyadenylation signal. Geneexpression is usually placed under the control of a promoter, and such astructural gene is said to be “operably linked to” the promoter.Similarly, a regulatory element and a core promoter are operably linkedif the regulatory element modulates the activity of the core promoter.

The terms “host cell”, “host cell line”, and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” or “transfectants” and “transformed cells”and “transfected cells” which include the primary transformed cell andprogeny derived therefrom without regard to the number of passages.Progeny may not be completely identical in nucleic acid content to aparent cell, but may contain mutations. Mutant progeny that have thesame function or biological activity as screened or selected for in theoriginally transformed cell are included herein.

The term “cell” includes both prokaryotic cells, which are used forpropagation of plasmids, and eukaryotic cells, which are used for theexpression of a nucleic acid. In one embodiment the eukaryotic cell is amammalian cell. In one embodiment the mammalian cell is selected fromthe group of mammalian cells comprising CHO cells (e.g. CHO K1, CHODG44), BHK cells, NS0 cells, Sp2/0 cells, HEK 293 cells, HEK 293 EBNAcells, PER.C6® cells, and COS cells.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues.

An “individual” or “subject” is a vertebrate. In one embodiment thevertebrate is a mammal. Mammals include, but are not limited to,domesticated animals (e.g., cows, sheep, cats, dogs, and horses),primates (e.g., humans and non-human primates such as monkeys), rabbits,and rodents (e.g., mice and rats). In certain embodiments, theindividual or subject is a human. In other embodiments the individual orsubject is a rabbit.

“Operably linked” refers to a juxtaposition of two or more components,wherein the components so described are in a relationship permittingthem to function in their intended manner. For example, a promoterand/or enhancer are operably linked to a coding sequence, if it acts incis to control or modulate the transcription of the linked sequence.Generally, but not necessarily, the DNA sequences that are “operablylinked” are contiguous and, where necessary to join two protein encodingregions such as a secretory leader and a polypeptide, contiguous and in(reading) frame. However, although an operably linked promoter isgenerally located upstream of the coding sequence, it is not necessarilycontiguous with it. Enhancers do not have to be contiguous. An enhanceris operably linked to a coding sequence if the enhancer increasestranscription of the coding sequence. Operably linked enhancers can belocated upstream, within or downstream of coding sequences and atconsiderable distance from the promoter. A polyadenylation site isoperably linked to a coding sequence if it is located at the downstreamend of the coding sequence such that transcription proceeds through thecoding sequence into the polyadenylation sequence. A translation stopcodon is operably linked to an exonic nucleic acid sequence if it islocated at the downstream end (3′ end) of the coding sequence such thattranslation proceeds through the coding sequence to the stop codon andis terminated there. Linking is accomplished by recombinant methodsknown in the art, e.g., using PCR methodology and/or by ligation atconvenient restriction sites. If convenient restriction sites do notexist, then synthetic oligonucleotide adaptors or linkers are used inaccord with conventional practice.

The term “peptide linker” denotes amino acid sequences of natural and/orsynthetic origin.

They consist of a linear amino acid chain wherein the 20 naturallyoccurring amino acids are the monomeric building blocks. The peptidelinker has a length of from 1 to 50 amino acids, in one embodimentbetween 1 and 28 amino acids, in a further embodiment between 2 and 25amino acids. The peptide linker may contain repetitive amino acidsequences or sequences of naturally occurring polypeptides. The linkerhas the function to ensure that polypeptides conjugated to each othercan perform their biological activity by allowing the polypeptides tofold correctly and to be presented properly. In one embodiment thepeptide linker is rich in glycine, glutamine, and/or serine residues.These residues are arranged e.g. in small repetitive units of up to fiveamino acids, such as GS (SEQ ID NO: 1), GGS (SEQ ID NO: 2), GGGS (SEQ IDNO: 3), and GGGGS (SEQ ID NO: 4). The small repetitive unit may berepeated for one to five times. At the amino- and/or carboxy-terminalends of the multimeric unit up to six additional arbitrary, naturallyoccurring amino acids may be added. Other synthetic peptidic linkers arecomposed of a single amino acid, which is repeated between 10 to 20times and may comprise at the amino- and/or carboxy-terminal end up tosix additional arbitrary, naturally occurring amino acids. All peptidiclinkers can be encoded by a nucleic acid molecule and therefore can berecombinantly expressed. As the linkers are themselves peptides, thepolypeptide connected by the linker are connected to the linker via apeptide bond that is formed between two amino acids.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, including digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

A “polypeptide” is a polymer consisting of amino acids joined by peptidebonds, whether produced naturally or synthetically. Polypeptides of lessthan about 25 amino acid residues may be referred to as “peptides”,whereas molecules consisting of two or more polypeptides or comprisingone polypeptide of more than 100 amino acid residues may be referred toas “proteins”. A polypeptide may also comprise non-amino acidcomponents, such as carbohydrate groups, metal ions, or carboxylic acidesters. The non-amino acid components may be added by the cell, in whichthe polypeptide is expressed, and may vary with the type of cell.Polypeptides are defined herein in terms of their amino acid backbonestructure or the nucleic acid encoding the same. Additions such ascarbohydrate groups are generally not specified, but may be presentnonetheless.

A “structural gene” denotes the region of a gene without a signalsequence, i.e. the coding region.

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindt,T. J., et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., N.Y.(2007), page 91) A single VH or VL domain may be sufficient to conferantigen-binding specificity. Furthermore, antibodies that bind aparticular antigen may be isolated using a VH or VL domain from anantibody that binds the antigen to screen a library of complementary VLor VH domains, respectively (see, e.g., Portolano, S., et al., J.Immunol. 150 (1993) 880-887; Clackson, T., et al., Nature 352 (1991)624-628).

The term “variant” denotes variants of a parent amino acid sequence thatcomprises one or more amino acid substitution, addition, or deletion.

The term “vector” denotes a nucleic acid molecule capable of propagatinganother nucleic acid to which it is linked. The term includes the vectoras a self-replicating nucleic acid structure as well as the vectorincorporated into the genome of a host cell into which it has beenintroduced. Certain vectors are capable of directing the expression ofnucleic acids to which they are operatively linked. Such vectors arereferred to herein as “expression vectors”.

General Steps of the Method as Reported Herein Immunization

Often non-human animals, such as mice, rabbits, hamster and rats, areused as animal model for evaluating antibody based therapies. Alsopossible is to use the B-cells of a human that survived a specificdisease, that suffers from a chronic disease, or that was recentlyvaccinated against a specific disease.

In the method as reported herein B-cells obtained from e.g. mouse, rat,hamster, rabbit, sheep, llama, or human can be used. In one embodimentthe mouse is an NMRI-mouse or a balb/c-mouse. In another embodiment thehamster is selected from Armenian hamster (Cricetulus migratorius),Chinese hamster (Cricetulus griseus), and Syrian hamster (Mesocricetulusauratus). In a specific embodiment the hamster is the Armenia hamster.In one embodiment the rabbit is selected from New Zealand White (NZW)rabbits, Zimmermann-rabbits (ZIKA), Alicia-mutant strain rabbits,basilea mutant strain rabbits, transgenic rabbits with a humanimmunoglobulin locus, rabbit IgM knock-out rabbits, and cross-breedingthereof.

In one embodiment the experimental animals, e.g. mice, hamsters, ratsand rabbits, chosen for immunization are not older than 12 weeks.

Source and Isolation of B-Cells

The blood of an experimental animal or a human provides a high diversityof antibody producing B-cells. The therefrom obtained B-cells secreteantibodies that have almost no identical or overlapping amino acidsequences within the CDRs, thus, show a high diversity.

In one embodiment the B-cells of an experimental animal or a human, e.g.from the blood, are obtained from 4 days after immunization until atleast 9 days after immunization or the most recent boost immunization.This time span allows for a high flexibility in the method as reportedherein. In this time span it is likely that the B-cells providing forthe most affine antibodies migrate from spleen to blood (see e.g. Paus,D., et al., JEM 203 (2006) 1081-1091; Smith, K. G. S., et al., The EMBOJ. 16 (1997) 2996-3006; Wrammert, J., et al., Nature 453 (2008)667-672).

B-cells from the blood of an experimental animal or human may beobtained with any method known to a person skilled in the art. Forexample, density gradient centrifugation (DGC) or red blood cell lysis(lysis) can be used. Density gradient centrifugation compared to lysisprovides for a higher overall yield, i.e. number of B-cell clones.Additionally from the cells obtained by density gradient centrifugationa larger number of cells divides and grows in the co-cultivation step.Also the concentration of secreted antibody is higher compared to cellsobtained with a different method. Therefore, in one embodiment theprovision of a population of B-cells is by density gradientcentrifugation.

Isolation of mRNA, Cloning, and Sequencing

From the B-cells the total mRNA can be isolated and transcribed to cDNA.With specific primers, the cognate VH- and VL-region encoding nucleicacids can be amplified. With the method as reported herein almost noidentical sequences will be obtained. Thus, the method provides forhighly diverse antibodies binding to the same antigen.

In one embodiment the methods as reported herein are for producing anantibody comprising cognate antibody variable domains. In one embodimentthe cognate antibody variable domains are from a single B-cell.

Primer can be provided for the amplification of the VH-encoding nucleicacid obtained from B-cells of the NMRI-mouse, the Armenian Hamster, theBalb/c-mouse, the Syrian hamster, the rabbit, the rat, the sheep, thellama, and the human.

One aspect as reported herein is a method for producing an antibodycomprising the following steps:

-   -   a) depositing single (mature) B-cells (obtained from the blood        or a lymphoid organ of an experimental animal or a human) from a        stained population of B-cells (in one embodiment the B-cells are        stained with one to three, or two to three fluorescence dyes) in        individual containers (in one embodiment is the container a well        of a multi well plate),    -   b) cultivating the deposited individual B-cells in the presence        of feeder cells and a feeder mix (in one embodiment the feeder        cells are EL-4 B5 cells, in one embodiment the feeder mix is        natural TSN (supernatant of a cultivation of thymocytes of an        experimental animal of the same species from which the B-cells        are derived), in one embodiment the feeder mix is a synthetic        feeder mix),    -   c) determining the amino acid sequence of the variable light and        heavy chain domains of specifically binding antibodies by a        reverse transcription PCR (RT-PCR) and nucleotide sequencing,        and thereby obtaining a monoclonal antibody variable light and        heavy chain domain encoding nucleic acid,    -   d) cultivating a cell comprising a nucleic acid encoding the        variable light and heavy chain in individual HC and LC        expression cassettes and recovering the antibody from the cell        or the cell culture supernatant and thereby producing an        antibody.

In one embodiment the method comprises the following steps:

-   -   a) providing a population of (mature) B-cells (obtained from the        blood or a lymphoid organ of an experimental animal or a human),    -   b) staining the cells of the population of B-cells with at least        one fluorescence dye (in one embodiment with one to three, or        two to three fluorescence dyes),    -   c) depositing single cells of the stained population of B-cells        in individual containers (in one embodiment is the container a        well of a multi well plate),    -   d) cultivating the deposited individual B-cells in the presence        of feeder cells and a feeder mix (in one embodiment the feeder        cells are EL-4 B5 cells, in one embodiment the feeder mix is        natural TSN (supernatant of a cultivation of thymocytes of an        experimental animal of the same species from which the B-cells        are derived), in one embodiment the feeder mix is a synthetic        feeder mix),    -   e) determining the binding specificity of the antibodies        secreted in the cultivation of the individual B-cells,    -   f) isolating the total RNA of a B-cell secreting an antibody        with the desired binding specificity,    -   g) performing with the polyA⁺ extracted mRNA an RT-PCR with        primer specific for the light and heavy chain variable domains,    -   h) determining the amino acid sequence of the variable light and        heavy chain domains of specifically binding antibodies,    -   i) introducing the monoclonal antibody light and heavy chain        variable domain encoding nucleic acids in respective expression        cassettes for the expression of an antibody,    -   j) introducing the nucleic acid into a cell,    -   k) cultivating the cell and recovering the antibody from the        cell or the cell culture supernatant and thereby producing an        antibody.

Specific Embodiments

In the method as reported herein the isolation of nucleic acid segmentsencoding antibody variable domains (light and heavy chain) and theinsertion of the isolated nucleic acid segments in eukaryotic expressionplasmids (one expression cassette each for the light and heavy chain,respectively) is performed without the intermediate isolation andanalysis of clonal intermediate plasmids. Thus, in the method asreported herein the intermediate cloning, isolation and analysis ofintermediate cassettes/plasmids is not required, e.g. by analysis ofisolated transformed E. coli cells. In one embodiment the methods asreported herein are performed without the intermediate isolation andanalysis of clonal intermediate plasmids.

It has been found that in the methods as reported herein the respectivenucleic acid fragments encoding the heavy and light chain variabledomain of an antibody as obtained after a specific polymerase chainreaction can be inserted into eukaryotic expression constructs, one foreach chain, respectively, and expanded without the requirement ofintermediate plating in order to pick and analyze plasmid DNA obtainedfrom single colonies of transformed bacteria.

Typically, a restriction endonuclease cleavage site is engineered intoboth sense and antisense primer, respectively, allowing the insertion ofthe PCR fragments into an appropriately designed expression vector.However, the relatively high promiscuity of the ligation process resultsin a comparatively high number of individual plasmid clones containingno inserted nucleic acid fragment (“empty vector”), containing a nucleicacid fragment inserted in the wrong direction, or containing only anincomplete fragment of the nucleic acid to be cloned. This problemusually is solved by plating the ligation reaction on solid media insuch a way that individual bacterial colonies can be isolated. Severalof these bacterial colonies (clones) are then picked and grown in liquidculture, and the respective plasmids contained in these clones areanalyzed for orientation and completeness of the inserted nucleic acidfragments. One of the correctly assembled plasmids is then selected andfurther amplified for e.g. the recombinant expression of the encodedpolypeptides. While being a multi time-tested method for the cloning ofa small, limited number of DNA fragments, this method is cumbersome,laborious and time-consuming when the cloning of a large number ofnucleic acid fragments is required because of the necessity to pick,amplify and analyze the plasmid DNA derived from single colonies asspecified above.

Thus, it has been found that the entire workflow from the initialgeneration and cloning of the DNA fragments into expression vectorsuntil the recombinant expression of the polypeptides encoded by therespective plasmid vectors can be performed in one coherent workflowwithout the need for intermediate isolation and analysis of singlecolonies. It has been found that it is advantageous to employligation-independent cloning as a means to improve the above-outlinedworkflow. Thus, in one embodiment the inserting in the eukaryoticexpression plasmid is by ligation-independent cloning.

Ligation-independent cloning as such is not necessarily more efficientthan conventional cloning via restriction and ligation in the sense thatthe number of individual colonies obtained is significantly higher. Butsince this method is based on sequence-specific annealing ofcomplementary single-stranded DNA overhangs rather than enzymaticligation for the assembly of complex molecules, this method comprisessignificantly longer complementary single-stranded ends of theindividual nucleic acid fragments to be cloned. Thus, typicallysingle-stranded nucleotide overhangs encompassing 15-30 nucleotides areused in ligation-independent cloning versus 2-4 nucleotides generated byrestriction endonucleases. In addition, since no ligase enzyme ispresent in ligation-independent cloning, the re-ligation of empty vectorcannot occur. Consequently, the proportion of correctly inserted nucleicacid fragments into a vector with regard to size, orientation andintegrity is increased while simultaneously the proportion of “empty”,re-ligated vector containing no inserted nucleic acid fragment at alland the frequency of plasmids containing defective DNA fragments aredecreased in ligation-independent cloning. Indeed, the analysis ofcloning products obtained by ligation-independent cloning showed thatover 90% of all plasmid molecules contained a full length insertednucleic acid in the correct orientation.

It has been found, since the vast majority of all plasmid molecules thusgenerated contains correctly inserted nucleic acid fragments, the entirepool of transformed bacteria can be grown and expanded in liquid culturewithout the need for the intermediate steps of plating the transformedbacteria on solid media, isolation of single colonies, and analysis andisolation of individual plasmid DNA clones. With the method as reportedherein a reduction in time and labor required can be achieved. With thisimproved method an automatization of the process can be performed.

The difference between the classical approach and the method as reportedherein is outlined in the following Table 1. It can be seen that thenumber of steps required can be reduced by more than 40%.

TABLE 1 classical approach ligation-independent approach generate andpurify PCR generate and purify PCR fragment fragment prepare vector forligation prepare vector for annealing restriction enzyme treatmentT4-DNA polymerase treatment purify DNA insert — ligation of fragmentwith annealing of fragment with prepared vector prepared vectortransformation into competent transformation into competent bacteriabacteria plate and grow on solid media grow in bulk in liquid culturepick individual colonies (clones) — grow clones in liquid culture —isolate plasmid DNA from colonies — analyze plasmid DNA from colonies —grow correct clone in liquid culture — isolate plasmid DNA isolateplasmid DNA transfect eukaryotic cells with expression transfecteukaryotic cells with plasmid expression plasmid — denotes: not neededto be performed.

The method as reported herein can be performed with B-cells obtained atany point in time after the immunization of an experimental animal.

The method as reported herein can be performed early after immunizationso that first antibody-encoding nucleic acids can be isolated as earlyas three weeks after the first immunization of an experimental animal.

The method as reported herein is especially suited for the isolation ofvariable domain-encoding nucleic acid fragments from rabbit B-cellssince hybridomas derived from rabbit B-cells result in poorly producingclones. In addition the isolation of variable domain-encoding nucleicacid fragments from rabbit-derived hybridomas is interfered by theendogenous light chain transcript of the commonly used myeloma fusionpartner.

The method as reported herein is faster compared to the classicalapproach.

In one embodiment the B-cell is a human B-cell, or a mouse B-cell, or arat B-cell, or a rabbit B-cell, or a hamster B-cell, or a B-cell derivedfrom a transgenic animal. In one embodiment the B-cell is a rabbitB-cell, or a human B-cell, or a B-cell derived from a transgenic animal.

A transgenic animal is an animal in which the endogenous Ig locus hasbeen inactivated or removed and which comprises an active or functionalhuman Ig locus.

In one embodiment the B-cell is a B-cell of an immunized experimentalanimal.

In one embodiment the B-cell is a B-cell of an immunized humanindividual, or a human individual that has survived a disease, or ahuman that is suffering from a chronic disease.

In one embodiment the B-cell is a single deposited antibody secretingB-cell.

In one embodiment the B-cell is cultivated for 6 to 8 generations.

In one embodiment the B-cell is cultivated until about 10 to about 100cells are obtained.

In one embodiment the B-cell produces about 10 ng/ml antibody after 7days of cultivation. In one embodiment the B-cell produces about 20ng/ml antibody after 7 days of cultivation.

It has been found, that if a B-cell producing less than 10 ng/mlantibody is used the method as reported herein can also be performed butwith lesser amplification efficiency.

In one embodiment the nucleic acid fragments encoding the variabledomains are isolated and/or amplified by RT-PCR.

In one embodiment the nucleic acid fragment encoding the variable lightchain domain and the nucleic acid fragment encoding the variable heavychain domain are cognate nucleic acids. In one embodiment the nucleicacid fragments encoding the heavy and light chain variable domains areisolated from the same cell and/or their progeny.

In one embodiment the B-cell is a rabbit B-cell and the nucleic acidencoding the variable heavy chain domain is isolated with the primer ofSEQ ID NO: 5 (AAGCTTGCCACCATGGAGACTGGGCTGCGCTGGCTTC) and SEQ ID NO: 6(CCATTGGTGAGGGTGCCCGAG).

In one embodiment the B-cell is a rabbit B-cell and the nucleic acidencoding the variable light chain domain is isolated with the primer ofSEQ ID NO: 7 (AAGCTTGCCACCATGGACAYGAGGGCCCCCACTC) and SEQ ID NO: 8(CAGAGTRCTGCTGAGGTTGTAGGTAC).

In analogy, primer for amplification of e.g. rat, mouse or humanimmunoglobulin V-domain gene segments can be designed. In one embodimentthe primer are directed to sequences in the first framework region. Seee.g. van Dongen, J. J. M., et al. Leukemia 17 (2003) 2257; Widhopf, G.F., et al. Blood 111 (2008) 3137; Fais, F., et al. J. Clin. Invest. 102(1998) 1515 for human B-cells; see e.g. Wang, Z., et al. J. Immunol.Methods. 233 (2000) 167; Jones, T. and Bendig, M., Bio/Technology 90(1991) 88 for murine B-cells.

In one embodiment the amplified nucleic acid is used withoutpurification after removal of the PCR primer.

In one embodiment the amplified nucleic acid is used withoutpurification after removal of all PCR primer.

In one embodiment the nucleic acid fragments encoding the variabledomains are inserted by sequence and ligation independent cloning (SLIC)into the eukaryotic expression plasmid.

In one embodiment the insertion does not require restriction enzymecleavage sites.

In one embodiment the insertion does not require a phosphatase treatmentof the nucleic acid fragment.

In one embodiment the integration does not require an enzymaticligation.

In one embodiment the T4 DNA polymerase is employed in the absence ofnucleotides for generating single strand extensions.

In one embodiment about 200 ng nucleic acid (=PCR product) is used inthe insertion step. In one embodiment about 100 ng nucleic acid is used.In one embodiment about 50 ng nucleic acid is used.

In one embodiment the ratio of plasmid to nucleic acid is about 1:2(w/w). In one embodiment about 100 ng plasmid and about 200 ng nucleicacid are used in the insertion step.

In one embodiment the method is a high throughput method.

In one embodiment the method is performed in parallel for at least tenB-cell clones.

In one embodiment the efficiency of the method starting from theamplification product to the recombinantly expressed antibody is morethan 50%.

It has been found that it is especially suited to employ in the methodas reported herein a pool of nucleic acids obtained from a pool of E.coli cells containing the assembled and/or amplified antibody expressionplasmids for the antibody light and heavy chain, respectively. Therewithpotential errors during the nucleic acid amplification of single clonescan be leveled, or masked, or reduced to background level.

In one embodiment the nucleic acid is a pool of nucleic acids obtainedfrom a pool of E. coli cells containing the assembled and/or amplifiedantibody expression plasmids for the antibody light and heavy chain,respectively.

It has been found that the PCR primers have to be removed or likewisethe PCR product has to be purified prior to the sequencing step. Thisseparation/purification increases the sequencing efficiency.

It has been found that it is advantageous to amplify the backbone of theplasmid excluding the nucleic acids encoding the variable domains.

In one embodiment the plasmid from which the vector (or plasmid)backbone is amplified is linearized prior to the amplification. In oneembodiment the plasmid is linearized by the use of two or more differentrestriction enzymes prior to the amplification.

In one embodiment the amplification product is digested with amethylation dependent restriction enzyme, e.g. DpnI.

This allows for more flexibility and efficiency in the subsequent methodsteps.

In one embodiment the method comprises the following steps:

-   -   total RNA extraction from antibody-producing B-cells of an        immunized experimental animal,    -   single stranded cDNA synthesis/reverse transcription of the        extracted polyA⁺ mRNA,    -   PCR with species specific primer,    -   removal of the PCR primer/purification of the PCR product,    -   optionally sequencing of the PCR product,    -   T4 polymerase incubation of the PCR product,    -   linearization and amplification of plasmid-DNA,    -   T4 polymerase incubation of the amplified plasmid-DNA,    -   sequence and ligation independent cloning of the variable domain        encoding nucleic acid into the amplified plasmid,    -   preparation of plasmid from pool of plasmid transformed E. coli        cells,    -   transfection of eukaryotic cells with plasmid prepared in the        previous step,    -   expression of antibody.

In one embodiment the light chain encoding plasmid backbone DNA isamplified with the primer of

SEQ ID NO: 9  (GTACCTACAACCTCAGCAGCACTCTG) and  SEQ ID NO: 10(CCCTCRTGTCCATGGTGGCAAGCTTCCTCTGTGTTCAGTGCT G).

In one embodiment the heavy chain encoding plasmid backbone DNA isamplified with the primer of SEQ ID NO: 11 (TGGGAACTCGGGCACCCTCACCAATGG)and SEQ ID NO: 12 (GCCCAGTCTCCATGGTGGCAAGCTTCCTCTGTGTTCAGT GCTG).

The following examples and sequence listing are provided to aid theunderstanding of the present invention, the true scope of which is setforth in the appended claims. It is understood that modifications can bemade in the procedures set forth without departing from the spirit ofthe invention.

Sequences:

SEQ ID NO: 1 linker peptide 1SEQ ID NO: 2 linker peptide 2SEQ ID NO: 3 linker peptide 3SEQ ID NO: 4 linker peptide 4SEQ ID NO: 5 heavy chain variable domain isolation primer 1(rb-VH3-23-Slic-s001 primer)SEQ ID NO: 6 heavy chain variable domain isolation primer 2 (rb-CH1rev-2primer)SEQ ID NO: 7 light chain variable domain isolation primer 1(rb-V-kappa-Slic-s001 primer)SEQ ID NO: 8 light chain variable domain isolation primer 2 (rbCk1-rev2primer)SEQ ID NO: 9 light chain plasmid amplification primer 1 (8011-Slic-s001primer)SEQ ID NO: 10 light chain plasmid amplification primer 2(8000-Slic-as002 primer)SEQ ID NO: 11 heavy chain plasmid amplification primer 1 (8001-Slic-s001primer)SEQ ID NO: 12 heavy chain plasmid amplification primer 2(8001-Slic-as002 primer)SEQ ID NO: 13 rb-V-kappa-HindIIIs primerSEQ ID NO: 14 rb-C-kappa-NheIas primerSEQ ID NO: 15 rb-CH1rev-1 primerSEQ ID NO: 16 rbVH3-23for3 primerSEQ ID NO: 17 bcPCR-huCgamma-rev primerSEQ ID NO: 18 bcPCR-FHLC-leader-fw primerSEQ ID NO: 19 bcPCR-huCkappa-rev primerSEQ ID NO: 20 bcPCR-hu-HC-10600-SLIC-as primerSEQ ID NO: 21 bcPCR-hu-HC-10600-SLIC-s primerSEQ ID NO: 22 bcPCR-hu-LC-10603-SLIC-s primerSEQ ID NO: 23 bcPCR-hu-LC-10603-SLIC-as primerSEQ ID NO: 24 SLIC-hu-VHuniversal-for primerSEQ ID NO: 25 SLIC-hu-VH6-for primerSEQ ID NO: 26 hu-CH1gamma-rev primerSEQ ID NO: 27 SLIC-huVk2-for primerSEQ ID NO: 28 SLIC-huVk3-for primerSEQ ID NO: 29 SLIC-huVk5-for primerSEQ ID NO: 30 SLIC-huVk7-for primerSEQ ID NO: 31 SLIC-huVk8-for primerSEQ ID NO: 32 SLIC-huVk1long-for primerSEQ ID NO: 33 SLIC-huVk2longw-for primerSEQ ID NO: 34 huCk-rev primerSEQ ID NO: 35 SLIC-huVl1-for primerSEQ ID NO: 36 SLIC-huVl2-for primerSEQ ID NO: 37 SLIC-huVl3-for primerSEQ ID NO: 38 SLIC-huVl4-for primerSEQ ID NO: 39 SLIC-huVl5-for primerSEQ ID NO: 40 SLIC-huVl6-for primerSEQ ID NO: 41 SLIC-huVl7-for primerSEQ ID NO: 42 SLIC-huVl8-for primerSEQ ID NO: 43 SLIC-huVl9-for primerSEQ ID NO: 44 SLIC-huVlambda10-for primerSEQ ID NO: 45 huC1-1-rev primerSEQ ID NO: 46 huIg-PCR-vectorprimer-asSEQ ID NO: 47 huIg-PCR-vectorprimer-VH-sSEQ ID NO: 48 huIg-PCR-vectorprimer-as kappaSEQ ID NO: 49 huIg-PCR-vectorprimer-VK-sSEQ ID NO: 50 huIg-PCR-vectorprimer-as lambdaSEQ ID NO: 51 huIg-PCR-vectorprimer-VL-s

EXAMPLES Example 1: Cloning and Expression of Cognate Antibody VariableRegion Gene Segments a) RNA Extraction

Cells were lysed by adding of 100 μl RLT buffer containing 10 μl/ml2-mercaptoethanol and mixing by repeated pipetting. The lysate waseither used directly for RNA isolation or stored frozen at −20° C. untilRNA preparation. RNA was prepared using the Total RNA Isolation KitNucleoSpin (Machery & Nagel) according to the manufacturer'sinstructions

b) First Strand cDNA Synthesis

cDNA was generated by reverse transcription of mRNA using the SuperScript III first-strand synthesis SuperMix (Invitrogen) according to themanufacturer's instructions. In a first step 6 μl of the isolated mRNAwas mixed with 1 μl annealing buffer and 1 μl (50 μM) oligo dT,incubated for 5 minutes at 65° C. and thereafter immediately placed onice for about 1 minute. Subsequently while still on ice 10 μl 2×First-Strand Reaction Mix and SuperScript™ III/RNaseOUT™ Enzyme Mix wereadded. After mixing the reaction was incubated for 50 minutes at 50° C.The reaction was terminated by incubation at 85° C. for 5 minutes. Aftertermination the reaction mix was placed on ice.

c) Polymerase Chain Reaction (PCR)

The polymerase chain reaction was carried out using AccuPrime PfxSuperMix (Invitrogen) according to the manufacturer's instructions.Light chain and heavy chain variable regions were amplified in separatereactions. PCR-primer (0.2 μM/reaction) with 25 bp overlaps to targetantibody expression vectors were used. After the PCR 8 μl of the PCRreaction mixture were used for analysis on 48-well eGels (Invitrogen).

d) Purification of PCR Products

Residual PCR primer were removed using the NucleoSpin® 96 Extract II kit(Machery & Nagel) according to the manufacturer's instructions.

e) Sequence Determination

The DNA sequences encoding the variable domains of the antibody heavyand light chains were obtained by sequencing the PCR products.

f) Preparation of Plasmid-DNA

The plasmid DNA to be used as recipient for the cloning of the PCRproducts encoding the antibody heavy and light chain variable domainswas first linearized by restriction enzyme digestion. Subsequently, thelinearized plasmid DNA was purified by preparative agaroseelectrophoresis and extracted from the gel (QIAquick Gel ExtractionKit/Qiagen). This purified plasmid DNA was added to a PCR-protocol astemplate using primer overlapping (by 20-25 bp) with the PCR-products tobe cloned. The PCR was carried out using AccuPrime Pfx SuperMix(Invitrogen).

g) Cloning

The PCR-products were cloned into expression vectors using a “site andligation independent cloning” method (SLIC) which was described by Haun,R. S., et al. (BioTechniques 13 (1992) 515-518) and Li, M. Z., et al.(Nature Methods 4 (2007) 251-256). Both purified vector and insert weretreated with 0.5 U T4 DNA polymerase (Roche Applied Sciences, Mannheim,Germany) per 1 μg DNA for 45 minutes at 25° C. in the absence of dNTPsto generate matching overhangs. The reaction was stopped by adding1/10^(th) of the reaction volume of a 10 mM dCTP Solution (Invitrogen).The T4 treated vector and insert DNA fragments were combined with aplasmid:insert ratio of 1:2 (w/w) (e.g. 100 ng:200 ng) and recombined byadding RecAProtein (New England Biolabs) and 10×RecA Buffer for 30minutes at 37° C. Subsequently, 5 μl of each of the generated heavychain and light chain expression plasmid was used to transform MultiShotStrip Well TOP 10 Chemically Competent E. coli cells (Invitrogen) usinga standard chemical transformation protocol. After regeneration (shakingfor 45 minutes at 37° C. of the transformed E. coli cells) the entiretransformation mixture was transferred into DWP 96 (deep well plates)containing 2 ml of LB medium supplemented with ampicillin per well. Thecells were incubated in a shaker for 20 hours at 37° C. In the followingstep the plasmid DNA encoding the immunoglobulin heavy- and light chainswas purified using the NucleoSpin 96 Plasmid Mini Kit (Macherey&Nagel),digested with selected restriction enzymes, and analyzed on 48-welleGels (Invitrogen). In parallel, glycerol stocks were prepared forstorage.

h) Transfection and Expression of Recombinant Antibodies in EukaryoticCells

HEK293 cells were grown with shaking at 120 rpm in F17-medium (Gibco) at37° C. in an atmosphere containing 8% CO₂. Cells were split the daybefore transfection and seeded at a density of 0.7-0.8×10⁶ cells/ml. Onthe day of transfection, 1-1.5×10⁶ HEK293 cells in a volume of 2 ml weretransfected with 0.5 μg HC plasmid plus 0.5 μg LC plasmid, suspended in1 μl 293-free medium (Novagen) and 80 μl OptiMEM® medium (Gibco) in 48well deep well plates. Cultures were incubated for 7 days at 180 rpm at37° C. and 8% CO₂. After 7 days the culture supernatants were harvested,filtered and analyzed for antibody content and specificity.

Example 2: B-Cell Productivity Vs. Amplification Efficiency

It has been found that B-cells to be used in the method as reportedherein have to be selected based on the expression yield (antibodytiter) obtained by the cultivation of the single deposited B-cell in thepresence of feeder cells, e.g. EL4-B5 cells and Zubler mix. The obtainedexpression yield has to be above a specific threshold value as can beseen from the following Table 2.

TABLE 2 rabbit IgG % HC % LC [ng/ml] sequences sequences Experiment 1<20 ng/ml 0% (0/14) 0% (0/14) Experiment 2 >20 ng/ml 85% (52/61) 85%(52/61)

Successful sequence generation depending on rabbit IgG concentration insingle cell cultivation supernatant.

Example 3: Primer Primer for B-Cell PCR of B-Cells Expressing RabbitAntibodies

Primer Set 1:

LC-Primer

-rb-V-kappa-HindIIIs (SEQ ID NO: 13): GATTAAGCTTATGGACAYGAGGGCCCCCACTC-rb-C-kappa-NheIas (SEQ ID NO: 14): GATCGCTAGCCCTGGCAGGCGTCTCRCTCTAACAG

HC-Primer

-rb-CH1rev-1 (SEQ ID NO: 15): GCAGGGGGCCAGTGGGAAGACTG-rbVH3-23for3 (SEQ ID NO: 16): CACCATGGAGACTGGGCTGCGCTGGCTTC

Primer Set 2:

LC-Primer

-rb-V-kappa-Slic-s001 (SEQ ID NO: 18):AAGCTTGCCACCATGGACAYGAGGGCCCCCACTC -rbCk1-rev2 (SEQ ID NO: 19):CAGAGTRCTGCTGAGGTTGTAGGTAC

HC-Primer

-rb-VH3-23-Slic-s001 (SEQ ID NO: 20):AAGCTTGCCACCATGGAGACTGGGCTGCGCTGGCTTC -rb-CH1rev-2 (SEQ ID NO: 21):CCATTGGTGAGGGTGCCCGAG

Primer for Amplification of Heavy Chain Expression Plasmid Backbone:

-8001-Slic-s001 (SEQ ID NO: 22): TGGGAACTCGGGCACCCTCACCAATGG-8001-Slic-as002 (SEQ ID NO: 23):GCCCAGTCTCCATGGTGGCAAGCTTCCTCTGTGTTCAGTGCTG

Primer for Amplification of Kappa Light Chain Expression PlasmidBackbone:

-8011-Slic-s001(SEQ ID NO: 24): GTACCTACAACCTCAGCAGCACTCTG-8000-Slic-as002(SEQ ID NO: 25):CCCTCRTGTCCATGGTGGCAAGCTTCCTCTGTGTTCAGTGCTGPrimer for B-Cell PCR of Rabbit B-Cells Expressing Human Antibodies(Derived from Transgenic Rabbit)

Primer for Amplification of Heavy Chain Variable Domains

HC-Up

-rb-VH3-23-Slic-s001(SEQ ID NO: 20):AAGCTTGCCACCATGGAGACTGGGCTGCGCTGGCTTC-bcPCR-huCgamma-rev (SEQ ID NO: 17): CCCCCAGAGGTGCTCTTGGA

Primer for Amplification of Light Chain Variable Domains

-bcPCR-FHLC-leader-fw (SEQ ID NO: 18): ATGGACATGAGGGTCCCCGC-bcPCR-huCkappa-rev (SEQ ID NO: 19): GATTTCAACTGCTCATCAGATGGC

Primer for the Amplification of Heavy Chain Plasmid Backbone:

-bcPCR-hu-HC-10600-SLIC-as (SEQ ID NO: 20):CAGCCCAGTCTCCATGGTGGCAAGCTTCCTCTGTGTTCAGTGCTG-bcPCR-hu-HC-10600-SLIC-s (SEQ ID NO: 21): CTCCAAGAGCACCTCTGGGGGCACAG

Primer for the Amplification of Kappa Light Chain Plasmid Backbone:

-bcPCR-hu-LC-10603-SLIC-s (SEQ ID NO: 22): GCCATCTGATGAGCAGTTGAAATC-bcPCR-hu-LC-10603-SLIC-as (SEQ ID NO: 23):GCGGGGACCCTCATGTCCATGGTGGCAAGCTTCCTCTGPrimer for B-Cell PCR of B-Cells from Human Donors

Primer for Amplification of Heavy Chain Variable Domains

-SLIC-hu-VHuniversal-for (SEQ ID NO: 24):AGCAACAGCTACAGGTGTGCATTCCGAGGTGCAGCTGKTGSAGTCTGS-SLIC-hu-VH6-for (SEQ ID NO: 25):AGCAACAGCTACAGGTGTGCATTCCCAGGTRCAGCTGCAGSAGTC-hu-CH1gamma-rev (SEQ ID NO: 26): GTCCACCTTGGTGTTGCTGGGCTT

Primer for Amplification of Kappa Light Chain Variable Domains

-SLIC-huVk2-for (SEQ ID NO: 27):TAGCAACAGCTACAGGTGTGCATTCCGATGTTGTGATGACTCAGTCT-SLIC-huVk3-for (SEQ ID NO: 28):TAGCAACAGCTACAGGTGTGCATTCCGAAATTGTGWTGACRCAGTCT-SLIC-huVk5-for (SEQ ID NO: 29):TAGCAACAGCTACAGGTGTGCATTCCGACATCGTGATGACCCAG-SLIC-huVk7-for (SEQ ID NO: 30):TAGCAACAGCTACAGGTGTGCATTCCGAAATTGTGCTGACTCAGTCT-SLIC-huVk8-for (SEQ ID NO: 31):TAGCAACAGCTACAGGTGTGCATTCCGAWRTTGTGMTGACKCAGTCTCC-SLIC-huVk1long-for (SEQ ID NO: 32):TAGCAACAGCTACAGGTGTGCATTCCGACATCCRGWTGACCCAGTCT-SLIC-huVk2longw-for (SEQ ID NO: 33):TAGCAACAGCTACAGGTGTGCATTCCGATRTTGTGATGACYCAGWCT-huCk-rev (SEQ ID NO: 34): ACACTCTCCCCTGTTGAAGCTC

Primer for Amplification of Lambda Light Chain Variable Domains

-SLIC-huV11-for (SEQ ID NO: 35):TAGCAACAGCTACAGGTGTGCATTCCCAGTCTGTGYTGACKCAG-SLIC-huV12-for (SEQ ID NO: 36):TAGCAACAGCTACAGGTGTGCATTCCCAGTCTGCCCTGACTCAG-SLIC-huV13-for (SEQ ID NO: 37):TAGCAACAGCTACAGGTGTGCATTCCTCCTATGAGCTGAYWCAG-SLIC-huV14-for (SEQ ID NO: 38):TAGCAACAGCTACAGGTGTGCATTCCCAGCYTGTGCTGACTCAA-SLIC-huV15-for (SEQ ID NO: 39):TAGCAACAGCTACAGGTGTGCATTCCCAGSCTGTGCTGACTCAG-SLIC-huV16-for (SEQ ID NO: 40):TAGCAACAGCTACAGGTGTGCATTCCAATTTTATGCTGACTCAG-SLIC-huV17-for (SEQ ID NO: 41):TAGCAACAGCTACAGGTGTGCATTCCCAGRCTGTGGTGACTCAG-SLIC-huV18-for (SEQ ID NO: 42):TAGCAACAGCTACAGGTGTGCATTCCCAGACTGTGGTGACCCAG-SLIC-huV19-for (SEQ ID NO: 43):TAGCAACAGCTACAGGTGTGCATTCCCWGCCTGTGCTGACTCAG-SLIC-huVlambda10-for (SEQ ID NO: 44):TAGCAACAGCTACAGGTGTGCATTCCCAGGCAGGGCTGACTCAG-huC1-1-rev (SEQ ID NO: 45): TCTCCACGGTGCTCCCTTC

Primer for Amplification of Human Immunoglobulin Expression Plasmid

Amplification of Heavy Chain Expression Plasmid Backbone:

-huIg-PCR-vectorprimer-as (SEQ ID NO: 46): GGAATGCACACCTGTAGCTGTTGCTA-huIg-PCR-vectorprimer-VH-s (SEQ ID NO: 47): AAGCCCAGCAACACCAAGGTGGAC

Amplification of Kappa Light Chain Expression Plasmid Backbone:

-huIg-PCR-vectorprimer-as kappa (SEQ ID NO: 48):GGAATGCACACCTGTAGCTGTTGCTA -huIg-PCR-vectorprimer-VK-s (SEQ ID NO: 49):GAGCTTCAACAGGGGAGAGTGT

Amplification of Lambda Light Chain Expression Plasmid Backbone:

-huIg-PCR-vectorprimer-as lambda (SEQ ID NO: 50):GGAATGCACACCTGTAGCTGTTGCTA -huIg-PCR-vectorprimer-VL-s (SEQ ID NO: 51):GAAGGGAGCACCGTGGAGA

Example 4: Pool Compared to Single Clones

Antibody variable region gene segments were amplified and cloned intothe respective expression vectors as described in Example 1. Todetermine the fidelity of sequences derived from pool-cloning versusconventional clone-picking from single colonies, the transformation mixwas split in two halves; one half was plated conventionally to generatesingle colonies while the other half was grown directly as pool-culture.Subsequently, plasmid was prepared both the pool-transformed E. colicells as well as from single colonies picked from the conventionalplates and the sequences of the cloned variable region gene segments wasdetermined. As shown in the following Table 3, between 80% and 100% ofcolony-derived plasmids contained a correct variable region gene segmentwhich was identical to the sequence obtained from thepool-transformation.

TABLE 3 Number of most abundant Number of most abundant B-cell LCsequence/total HC sequence/total Clone No. number of sequences number ofsequences 5 10/11  8/12 6 11/12 12/12 10 2/2  8/12 35 6/6  7/12 38 11/1212/12 39  9/11 10/12 42 3/5 11/12 50 6/6 6/7

The VH and VL encoding nucleic acid sequence obtained from thesequencing of the pool cultivated cells were identical to the mostabundant sequence obtained from the sequencing of individual clones.

1-15. (canceled)
 16. A method for producing an antibody, the methodcomprising: (i) obtaining an antibody-secreting B-cell from an animal,wherein the B-cell comprises nucleic acid encoding the antibody; (ii)culturing the B-cell as a single-deposited B-cell, wherein the B-celland its progeny produce greater than 20 ng/ml of the antibody in 7 daysof co-cultivation of the B-cell with feeder cells; (iii) amplifying thenucleic acid encoding the antibody light chain variable domain or theantibody heavy chain variable domain by PCR using single-stranded cDNAobtained from the RNA of the antibody-secreting B-cell as template,thereby obtaining a pool of nucleic acids encoding the antibody lightchain variable domain or the antibody heavy chain variable domain; (iv)generating single strand extensions of the pool of nucleic acids in theabsence of nucleotides using T4 DNA polymerase; and (v) inserting theamplified nucleic acids encoding the antibody light chain variabledomain or the antibody heavy chain variable domain into a eukaryoticexpression plasmid by sequence- and ligation-independent cloning,wherein the pool of nucleic acids encoding the antibody light chainvariable domain and the antibody heavy chain variable domain,respectively, is inserted into the expression plasmid.
 17. The method ofclaim 16, wherein the B-cell is a rabbit B-cell.
 18. The method of claim16 or claim 17, wherein the feeder cells are EL4-B5 cells.
 19. Themethod of claim 16 or claim 17, wherein PCR primers comprising thenucleic acid sequences of SEQ ID NOs:5 and 6 or the nucleic acidsequences of SEQ ID NOs:7 or 8 are used for the amplification of nucleicacid encoding the antibody light chain variable domain or the antibodyheavy chain variable domain, respectively.
 20. The method of claim 16 orclaim 17, wherein method further comprises removing PCR primers afterthe PCR amplification step.
 21. The method of claim 16 or claim 17,wherein about 300 ng nucleic acid is used in the insertion reaction.